First detection of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Ixodes ricinus ticks (Acari: Ixodidae) from multiple locations in Hungary

The parasitoid wasp, Ixodiphagus hookeri (Hymenoptera: Encyrtidae), is the natural enemy of a wide range of hard and soft tick species. While these encyrtid wasps are supposed to be distributed worldwide, only a few studies report on their actual distribution around the globe. Within a shotgun sequencing-based metagenome analysis, the occurrence of I. hookeri was screened at multiple Ixodes ricinus (Acari: Ixodidae) tick sampling points in Hungary to contribute to the assessment of the distribution patterns of the parasitoid wasps in Central Europe. To our knowledge, the first report of the species in Hungary and the description of the southernmost I. hookeri associated geoposition in Central Europe took place within our study. I. hookeri infested I. ricinus nymphs were detected at five sampling points in Hungary. The results show that the exact distribution range of I. hookeri is still barely studied. At the same time, unprecedented public health issues being brought about by climate change might require steps toward the exploitation of the tick biocontrol potential and as an ecological bioindicator role of the parasitoid wasp in the future.

www.nature.com/scientificreports/ As climate change, accompanied by various sociodemographic alterations, brings unprecedented challenges related to vector-borne diseases 12,13 , the need for the development of control methods against tick populations is a public highlight. Several methods have been introduced to address this issue. These control methods often rest on either conventional chemical acaricides or on further alternatives, such as biological control methods assisted by the natural enemies of ticks [14][15][16][17][18] . A line of biological control methods against ticks could be the Encyrtidae family members which are small-sized, parasitoid, or hyperparasitoid wasps distributed all around the globe. Due to their efficacy and target specificity, numerous wasps from this family are used as biological pest control, while several additional encyrtid species are documented as promising candidates for this role [19][20][21] . Ixodiphagus spp., including I. hookeri 22 , are encyrtid wasps attacking a wide range of tick species that have received relatively much attention as a specific and effective, natural alternative for biological hard tick control 23,24 . Interestingly, I. hookeri appears to have alternating preferences for the tick species and developmental stage of its hosts at geographically distant locations 25,26 . In European settings, Ixodiphagus wasps are described to parasitize the larvae and nymphs of hard ticks with a clear predilection for unfed nymphs 24 . If oviposition occurs in larvae, transstadial transmission through the molting of the ticks to nymphs can also occur 27 . Wasp eggs start their embryonic development in engorging or engorged nymphs. Wasp larvae feed on tick tissues and emerge as fully grown adults causing the death of the host before it can reach the adult stage 23,24 .
Ixodiphagus wasps have been associated with several hard and soft tick genera belonging to the families of Ixodidae and Argasidae, including Ornithodoros, Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus 23 . Studies conducted in Europe revealed that I. ricinus appears to be the preferred species of the European I. hookeri, while another common tick species, Dermacentor reticulatus is supposed not to be chosen as a host by the European representatives of the parasitoid wasps 24 .
While Ixodiphagus spp. have been detected in many countries and in a diverse range of hard and soft tick species, parasitoid wasps have been less studied in Hungary despite their potential to reduce tick populations and tick-borne disease cases. In the present study, our aim was to confirm the presence I. hookeri in a diverse set of locations in Hungary using a modern, sensitive metagenomic approach 28,29 . Due to their high density in Hungary, high public health significance as TBD transmitters, and potential to host Ixodiphagus wasps, I. ricinus ticks were decided to be assessed for the parasitoids. Based on our approach, genomic information of the European populations of I. hookeri may also be obtained, which can serve as a reference for further studies.
The blackPREP Tick DNA/RNA Kit (Analytik Jena GmbH) was used for the DNA isolation. Isolated total metagenome DNA was used for library preparation. In vitro fragment libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Paired-end fragment reads were generated on an Illumina NextSeq sequencer using TG NextSeq 500/550 High Output Kit v2 (300 cycles). Primary data analysis (base-calling) was carried out with Bbcl2fastq software (v2.17.1.14, Illumina).
On the raw sequencing data, quality-based filtering and trimming were performed by TrimGalore (v.0.6.6, https:// github. com/ Felix Krueg er/ TrimG alore), setting 20 as a quality threshold, retaining reads longer than 50 bp only. Using the remained reads, a de novo assembly was performed by MEGAHIT (v1.2.9) 32 using default settings. The resulting contigs were taxonomically classified using Kraken2 (k = 35) 33 with the NCBI non-redundant nucleotide database 34 . Contigs were predicted as I. hookeri by taxon classification and were checked with BLAST 35 on the partial sequence of I. hookeri 28S ribosomal RNA gene (MH077537.1) as a reference. Multiple sequence alignment was done by MAFFT (v7.490) 36 . All data management procedures, analyses, and plottings 37,38 were performed in the R environment (v4.2.1) 39 .

Results
Of the 21 adult female samples (10 individuals per sample) examined, we did not find any contigs with reasonable evidence for I. hookeri origin. In five of the 21 nymph samples (10 individual nymphs per sample), namely sample b, c, d, g, n, contigs deriving from I. hookeri were found. The sequence identity of the contigs deposited in GenBank to the I. hookeri 28S rRNA gene was 378/386 (97.9%), 556/559 (99.4%), 439/447 (98.2%), 445/453 (98.2%), and 300/308 (97.4%) for samples b (accession id: OQ316579), c (OQ316577), d (OQ316581), g (OQ316578), and n (OQ312115), respectively. The multiple sequence alignments of the contigs with the partial reference sequence of I. hookeri 28S rRNA gene (MH077537.1) are shown in Fig. 1.The figure shows that the sequence of the generated contigs varies from the reference sequence at 8 positions (A434T, A491C, A499G, A666G, A670G, T677G, G678T, C688G). In the annotation of the altered positions, the first letter refers to the base in the reference sequence, the following numbers specify the genomic position of the polymorphism, and the last letter indicates the base detected in our samples. By position A434T, transversions were identified in sample b, c, d, and g. All our samples included A491C and A499G mutations. A666G, A670G, T677G, G678T, and C688G polymorphisms occurred in sample b, d, g, and n. In the easternmost sample (n), we found one position www.nature.com/scientificreports/ (C565T) that differs from all other Hungarian samples and the reference sequence as well. The geoposition of the samples is presented in Fig. 2.

Discussion
The findings that no reads deriving from I. hookeri were detected in adult I. ricinus samples collected between the end of March and the middle of May, while nymphs were associated with this species, promote former theories of the life cycle of the parasitoid wasps 23,24 . I. hookeri eggs may have been laid in the larvae or in nymphs before winter or, less probably, during spring, as the wasps were formerly associated with the possibility of surviving winter conditions 40,41 .
To our knowledge, this is the first report on evidence of the presence of Ixodiphagus wasps, namely I. hookeri in Hungary. This finding expands the localities associated with I. hookeri within Europe. All except one sampling   54 and Ukraine (detected in the former Soviet Union) 55 . To our knowledge, sampling point d (Sáska) in Hungary represents the southernmost detection point of I. hookeri within Central Europe. The detection of I. hookeri in Hungary may serve as a novel hint regarding the potential distribution I. hookeri at the Balkan Peninsula, where the species appears to be little studied.
As mentioned above, wasp-negative sampling points can be wasp-invaded. Even though next-generation sequencing (NGS) based metagenomic approach appears to be just as or even more sensitive as polymerase chain reaction (PCR) based target detection techniques 28,29,56 , certain limitations can be addressed. Within the pool of reads deriving from the shotgun-sequenced metagenome that contains genome fragments from every organism present in the sample, lower relative abundance rates of an individual species serve with relatively lower read counts from its genome. In other words, shotgun sequencing preserves the original relative read abundance rates of the various organisms of the samples and may represent fewer reads of certain species by non-targeted runs 57,58 . Moreover, the I. hookeri reference sequence, that the metagenomic read sets were aligned to only represented a smaller fragment, namely the unique 28S ribosomal RNA gene of the full I. hookeri genome. Thus only I. hookeri reads deriving from this part of the genome could have been aligned, that further increases the chance of false-negative sampling points for the wasps. Besides the above-mentioned reasons, due to the low European tick parasitization rates of I. hookeri, the 10 nymphs collected at the sampling points may miss the wasps by chance alone.
Nevertheless, NGS-based approaches have a prospering future within the studies of parasitoids of public health significance, such as I. hookeri. According to Collatz and colleagues, large geographical distance and climatic differences (e.g. presence in Africa, Asia, Europe, and North America) 24,25,59,60 may even underlie divergence and distinct taxonomic categorization of I. hookeri to different species, subspecies or at least strains 24 . Concurrently, publications on I. hookeri indicate a certain extent of behavioral and host preferences at different www.nature.com/scientificreports/ continents 24,25,61 . To assess the basic variation in behavioral traits of I. hookeri or to identify specific characteristics of subgroups that can be better utilized by the biological control methods, the study of the I. hookeri genome or at least specific genome regions, such as 28S rRNA or 16S rRNA genes, may become inevitable, similarly to other weighty insect groups 52,62,63 . The improvement of our knowledge of I. hookeri with either traditional or genomic methods could facilitate the assessment of its potential as a means of biological control, while limitations and doubts about the wasps' biocontrol potential could be addressed with more research. Attempted mass releases of the parasitoid wasps in the U.S and in the former Soviet Union between 1920 and 1940 40,64,65 were unsuccessful as far as causing noticeable reduction of tick populations. One reason for this may be that I. hookeri requires high tick host densities and superabundant tick populations to reach its ideal abundance 61,66 . Inadequate numbers of parasitoids released compared to the geographical areas may have also undermined these trials 23 . On the other hand, the parasitoid wasps have been transported to the sites of attempted mass releases from great distances, sometimes even from different continents (e.g. from France to the U.S.) 40,64 without any considerations regarding their host preferences, climatic adaptations, or behavioral attributes, that have, since then turned out to be rather specific to their geographic locations of origin 24,25,61 . In a global perspective, climate-associated occurrence rate alterations 23,25 or differences in other characteristics of I. hookeri, such as the duration of its development, may also be underlain by host-related factors. Synchronization with the maturation of the tick host and, indirectly, with the main activity period of the vertebrate hosts of the ticks throughout the year may play a role in the life cycle of the wasps 24 .
Furthermore, we do not know how great the tick populations would be without the endemic I. hookeri populations and how much the parasitoid wasps contribute to maintaining the equilibrium of the communities in which they are included 67 . Nonetheless, the hypothesis regarding sufficiently high tick host densities and superabundant parasitoid host populations is in line with findings regarding the bioindication potential of certain insect species, including parasitoid wasps 68 . If so, this potential may also be worth further observation.
Conclusively, assessment of existing populations and further examinations on entomologic and genomic traits along with ecological roles could help understand and exploit the Ixodiphagus wasps' potential as a biological tick control method or as a potential bioindicator species.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.